Cinnamyl phenols useful as anti-microbial agents

ABSTRACT

SUBSTANCES WHICH ARE SUBJECT TO MICROBIAL SPOLAGE ARE PRESERVED BY ADDITION OF A CINNAMYL PHENOL, E.G., 2-CINNAMYL-PHEONOL, 4-CINNAMYL PHENOL, 2-METHOXY-4-CINNAMYLPHENOL, 2-CINNAMYL-5-METHOXY-QUNOL, ETC.

United States Patent Int. Cl. A0111 9/26 US. Cl. 424--341 3 Claims ABSTRACT OF THE DISCLOSURE Substances which are subject to microbial spoilage are preserved by addition of a cinnamyl phenol, e.g., 2-cinnamy1-phenol, 4-cinnamyl phenol, 2-methoXy-4-cinnamylphenol, Z-cinnarnyl-S-methoxy-quinol, etc.

This is a division of our co-pending application, Ser. No. 74,485, filed Sept. 22, 1970, now U .8. Pat. 3,745,222, issued July 10, 1973.

A non-exclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublioenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to the preservation of substances which are normally subject to microbial spoilage. The objects of the invention include the provision of novel processes and compositions for accomplishing such preservation. Further objects of the invention will be evident from the following disclosure wherein parts and percentages are by weight unless otherwise specified. The abbreviation p.p.m. used herein refers to parts per million. Temperatures are given in degrees centigrade. The symbol is used herein to designate the phenyl radical.

In accordance with the invention it has been found that certain agents exhibit unexpected antimicrobial activity and are useful for preserving all kinds of materials which are normally subject to microbial spoilage. The agents in question are certain cinnamyl-phenols, and their antimicrobial activity is unusual and unexpected because it is not shared by. closely-related phenol derivatives.

Generically, the agents in accordance with the invention have the structure:

where at least one but not more than three of the Rs are hydroxy radicals, and the remainder of the R are each independently selected from the group consisting of lower alkyl, lower alkoxy, and hydrogen.

The compounds of the invention are especially useful because they are active against many microorganisms in the categories of bacteria, yeasts, and molds. In other words, the compounds are not just active against one or a few organisms; rather, they display broad-spectrum antimicrobial activity.

Examples of particular compounds within the scope of the invention are given below by way of illustration and not limitation:

ice

4-cinnamyl-phenol (also known as obtusastyrene) 0H ;H1GH=CH- Z-cinnamyl-phenol @Hr-C 11:0 11- 2-cinnamyl-4-methyl-phenol 0H @Hz-CH IJH-4:

2-cinnamyl-4-methoxy-phenol 2-methyl-4-cinnamyl-phenol Hz-C H=C H-- 2-methoxy-4-cinnamyl-phenol 4-cinnamyl-resorcinol Z-cinnamyl-S-methoxy-quinol 4-cinnamyl-pyrogallol 3 The compounds of the invention display activities which are equal or even superior to those of widely-used antimicrobial agents. This is illustrated by the following: In general, the compounds of the invention are superior to such known agents as phenol, resorcinol, o-phenyl-phenol, and the alkali metal sorbates and benzoates. For instance, compounds of the invention, at concentrations of 12 to 25 p.p.m., were found to inhibit the growth of four bacteria (Bacillus cereus, Sarcina lutea, Staphylococcus aureus, and Streptococcus lactis) whereas to achieve the same effect it required 100 to 200 p.p.m. of o-phenylphenol, more than 800 p.p.m. of potassium sorbate, and more than 1000 p.p.m. of sodium benzoate.

Alkyl 4-hydroxybenzoates are well known to exhibit potent microbial activity. In general, the compounds of the invention display a superior activity a compared to these benzoates wherein the alkyl group contains less than seven carbon atoms. For instance, whereas the compounds of the invention at a concentration of 12 to 25 p.p.m. will inhibit the growth of the four bacteria noted above, it requires concentrations of 100 to 400 p.p.m. for the same result to be achieved with the C -C alkyl 4-hydroxybenzoates. Moreover, it may be noted that the compounds of the invention have an advantage in that their watersolubility is higher than that of the higher alkyl (e.g., heptyl) 4-hydroxybenzoates, and that the compounds of the invention do not exhibit the strong and unpleasant odor which characterizes the said benzoates. Also, with respect to some microorganisms, the compounds of the invention inhibit growth when applied at levels less than required with the higher alkyl 4-hydroxybenzoates. This is the case, for example, with bacteria such as Acaligenes faecelis and E. coli, yeasts such as Pichiw chodati, Hansenula anomala, and Saccharomyces cerevisiae, and molds including Aspergillus flavus, A. niger, Penicillium chrysogenum, Rhizopus semi, Botrytis cinerea, Byssochlamys fulva, and Alternaria sp.

Of the various compounds included within the scope of the invention, the cinnamyl-substituted monophenols display particularly high antibiotic activity against a large variety of different microorganisms, and therefore are preferred with respect to the cinnamyl derivatives of dior tri-phenols. Coming into special consideration is 4- cinnamyl-phenol because it displays antibiotic activity over a wide range of pH. The cinnamyl derivatives of monophenols are also preferred because they are essentially colorless, whereas those derived from dior triphenols exhibit various shades varying from red to brown.

As evident from the explanation immediately following Formula I above, the invention includes compounds wherein there may be nuclear lower alkyl or lower alkoxy substituents in addition to the hydroxy groups. In general, the compounds are preferred wherein such additional substituents are absent or, if present, are in small number, e.g., a total of one to two lower alkyl and/or lower alkoxy substituents. Taking the foregoing facts into consideration, we prefer to employ the compounds of the sub-generic category represented by the formula:

wherein R is lower alkyl,

R" is lower alkoxy,

n is an integer from 0 to 2,

m is an integer from 0 to 2, and

the sum of n and m is not more than 2.

The invention encompasses not only the use of any of the above-described agents individually, but also mixtures thereof.

In preserving substances in accordance with the invention, any of the aforesaid agents or mixtures thereof are incorporated with the substance, using an amount of the agent to inhibit microbial growth. Additional conventional treatments such as dehydration, canning, refrigeration, or freezing may be applied to the substance containing the added agent. The incorporation of the agent with the substance may involve a mixing of the substance and the agent-this is especially suitable where the substance is in liquid or particulate form. Where the substance is in the form of pieces of large dimensions the agent may be incorporated therewith by coating it on the surface of the pieces. For such purpose the agent is preferably dispersed in a carrier-a liquid such as water, alcohol, wateralcohol blends, oils, or a finely-divided solid such as salt, starch, talc, or the like.

The invention is of wide versatility and can be applied for the preservation of all kinds of substances which are normally subject to microbial spoilage. Typical examples of such substances are listed below by way of example. Foodstuffs such as fruits, vegetables, juices, milk, eggs, meat, fish, grains, cereal products, cheese, etc. Animal glues and mucilages; dextrins; starch pastes and solutions; cosmetic, medicinal, and dental preparations; vitamin preparations; pastes, solutions, or other preparations of natural gums such as tragacanth, arabic, acacia, karaya, locust bean, agar-agar, pectin, elgin, etc.; fermentation broths, mashes, and residues from fermentation processes; whey; wines and Vinegars; animal feeds and ingredients of animal feeds such as fish meals, blood meals, feather meal, meat scraps, bone meal, tankage, grains, and oil-seed meals; proteins and protein hydrolysates; textile printing pastes; paints containing proteins or other spoilable dispersing agents; solutions of bark extracts or other tanning agents; molasses; by-products or wastes that contain potentially valuable carbohydrate, proteinous or fat ingredients such as stick liquor, corn steep liquor, fruit cannery wastes, citrus peels, cull fruit and vegetables, tops of root vegetables, distillers slops, pulp liquors, Wash water from textile de-sizing operations, waste liquors from wool scouring plants, dairy and slaughter house wastes and liquors, etc.

The compounds of the invention may be synthesized by known proceduresfor example, those disclosed by Hurd et al., J our. Am. Chem., Soc., vol. 59, pp. 107-109; Barnes et al., Tetrahedron, vol. 21, pp. 2707-2715; Jurd, Experimentia, vol. 24, pp. 858-860; Jurd, Tetrahedron, vol. 25, pp. 1407-1416; and Jurd, Tetrahedron Letters No. 33, pp. 2863-2866. Typically, these syntheses involve condensing a selected phenol (phenol itself, a cresol, resorcinol, guaiacol, hydroquinone monomethyl ether, etc.) with cinnamyl bromide or cinnamyl alcohol.

The invention is further demonstrated by the following illustrative examples. For comparative purposes, various compounds, including some of known antimicrobial activity, are included in the reported experiments.

EX MPLE 1 A series of compounds were assayed for efiectiveness against bacteria, molds, and yeasts, using the following test procedure.

All compounds were tested at a concentration of 500 p.p.m. (w./v.). Plates were prepared by adding a measured amount of the candidate compound (in an appropristrips? The plates were incubated at 28 C. for one to five days and evaluated by comparison with the controls.

The results are expressed on the basis:

+ for eflfective to inhibit growth :t for not completely efiective; faint growth occurs for ineifective; growth occurs.

The test results are summarized in the following tables.

TABLE I Activity of Various Compounds (at concentration of 500 ppm) Against Bacteria 2-cinn- 2-cinn- 4-cinnamylamy am 1-5- amyl-o- Cinn- Methamylresorpyromet oxymethoxy amyl- R esor- Pyrooxyphenol einol galloi quinol qulnone eugenol Phenol cinol gallol quinol Bacillus cereus Sarcina lutea Staphylococcus aur Streptococcus lactta Acaliaenes faecalis. Escherichia coli. i Pacudomcmes aer'uginosa.-- Salmonella tuphimufium... d: Serratia marcescens i 5:

TABLE II Activity of Various Compounds (at concentration of 500 ppm.) Against Yeasts 4-cinn- 4-cinn- Z-cinn- 2-einn- 4-cinnamylnmylamyl-E- amyl-E- Cinn- M th. amylresorpyro methoxymethoxy amyl- Resor- Pyrooxyphenol cinol gallol quinol quinone eugenol Phenol einol gailol quinoi Zwosacchomuceajaprmicm. Candida tropicalis Pichia chodati Hammula anomala a cereviaiae i i Candida chalmerat. Saccharomyces rose Zwoaaccharo'myces barkeri TABLE III Activity oi Various Compounds (at concentration of 500 ppm.) Against Molds 4-cinn- 4-cinn- 2-cinn- 2-cinni-cinm amylamylamyl-5- amyl-5- Clnn- Methemylresor- PY phenol cinol gallol Asjlergillua flavua...-;-;..'. Asperaillus niaer Penicilliu'm chrwogenum.. i

Resor- Pyrooxymetho methoxy amylxy eugenol Phenol cinol galioi quinone l-i-Illll EXAMPLE 2 TABLE IV Minimal Inhibitory Concentrations (in p.p.m.) of Various Compounds Against Bacteria 2-cinn- 2-methyl- 2-einni-einn- 2-einnamyl-4- e-cinnamyl-4- Orotho- Potasamylamylmethoxynmylmethylphenylslum Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Bacillus cereus 25 12 25 12 12 100-200 800 1, 0o 0 Sarcma lutea- 25 25 25 12 12 200 800 1, 000 Staphylococcus a 25 25 25 12 12 200 800 1, 000 Streptococcus lactic" 25 12 25 12 12 100-200 800 1, 000 Acaligema faecalisn. 50-100 200 200 200 200 100-200 800 1, 000 Escherichia coli 50-100 200 200 200 200 100-200 800 1, 000

TABLE V Minimal Inhibitory Concentrations (in p.p.m.) of Various Compounds Against Yeasts 2-einn- 2-methy1- 2-cinn- 4-clnn- 2-cinnamyi-4- -cinnamyl-4- Potasamylamylmethoxyamylmethylo-Phenylslum Sodium phenol phenol phenol phenol phenol phenol 'sorbate henzoate Zygosaccharomyces jopom'cus 12 12 25 12 12 100-200 800 1. 000 Candida tropicalis 12 12 50 25 25 100 250-500 Pichia chodati- 25 50 200 200 200 100 800 1,000 Hansenula anomala. 50 50 100 100 50 100-200 800 1, 000 Saccharomycee cereoisiae 25 25 50 25 25 100-200 800 1,000 Torula utilis 50 50 200 100 200 100-200 800 1, 000

TABLE VI Minimal Inhibitory Concentrations (im p.p.m.) Various Compounds Against Molds 2-cinn- 2-methyl- 2-cinn- 4-cinn- 2-cinnamyl-4- a-cinnamyl-iv Potasamylamylmethoxyamylmethylo-Phenylslum Sodium phenol phenol phenol phenol phenol phenol sorbate benzoate Aspergillus flavus... 100 100 200 200 200 100 800 1, 000 Aspergillua m'ocr- 50 100 200 200 200 100 800 1, 000 Penicillium chm so 1mm 50 25-50 200 50 100 50 800 000 Rhizopus semi 6 50 200 50 100 100 200 500 Botn/tis cinerea- 25-50 12-25 200 50 25 12-25 200 500 Byssochlamys fu 25 25 50 25 25 50 800 1, 000 Alteruaria 8p..- 25 25 100 50 100 50 400 TABLE VII Minimal Inhibitory Concentrations (in p.p.m.) of 4-Cinnam3l-phenol and Several Akyl e-Hydroxybenzoates Against Bacteria, Yeasts, and Molds n-Butyl n-Propyl 4-einn- Heptyl 4- Amyl 4- 4.- 4- Ethyl 4- Methyl 4- amylhydroxyhydroxyhydroxyhydroxyhydroxyhydroxyphenol benzoate benzoate benzoate benzoate benzoate henzoa' te Bacillus cereus 25 12 100 200 400 200 200 Sarciua Zutea... 25 12 100 200 400 200 200 Staphylococcus aureus 25 12 100 200 400 200 200 Streptococcus lactis. 25 12 100 200 400 200 200 Acaligeucs faecalis 50-100 200 200 200 400 200 200 Escherichia coli 50-100 200 200 200 400 200 200 12 12-25 50 100 100-200 200 200 12 12-25 50 100 200 200 200 25 200 100 100 200-.400 200 200 50 200 100 100 200-400 200 200 25 100 50 100 200 200 200 50 25 100 100 200 200 200 Aspergillus flavus. 100 200 100 200 200 200 200 Aapcrgillus m'qer 50 200 100 200 200 200 200 Peuicillium chrysogeuu 50 200 100 100 200 200 200 Rhizopus semi 6 25 50 100 200 200 200 Botrytis cinerea... 25-50 50-100 50 100 100-200 100 100 Byssochlamys fulva. 25 2 100 100 200 200 Alternaria 3p 25 50-100 50 50 100-200 100 200 EXAMPLE 3 EXAMPLE 4 Two compounds (4-cinnamyl-phenol and 4-cinnamylresorcinol) were assayed for effectiveness against bacteria, molds, and yeasts, except that in this case the assays were conducted at different concentrations of each compound and at different pHs in order to determine the minimum concentration of each compound required to inhibit growth at the particular pH.

The results are tabulated below.

Samples of fruit juice (grape and apple) with and without added 4-cinnamy1-phenol were inoculated with cultures of wild yeast or pure wine yeast (S. cerevisiae) and held at room temperature. The inoculated juices were observed at intervals to detect the time at which fermentation (gas production) began. This is a. measure of microbial' activity-where the organisms multiply freely, the time for initiation of fermentation is short; converse- TABLE VIII Eflect of pH on Minimal Inhibitory Concentration (p.p.m.) of 4-Cinnamyl-phenol and 4-Cinnamyl-resorcino 4-cinnamyl-phenol, 4-cinnamyl-resorclnol,

pH ofpH Bacillus cereus 25 25 12.5 200 200 100 6.25 Sarciua lutea 25 25 Staphylococcus our 25 50 Streptococcus Zactia- 25 50 Acal' 50 100 Zygosaccharomyces japo'hicu 50 50 50 Candida tropicalis 50 50 50 .Pz'chia chodati. 150 100 50 Hansenula auo'mal 100 100 Saccharomz cea cereoisia 50 50 Geotrichum s Torula utilia 50 50 37.5 25 50 75 50 Asperoillus flavus..- 100 100 100 50 200 200 100 Aspergz'llus 'm'oer 100 100 75 37.5 200 100 100 Peuiccilliu'm chrysooeuum- 50 50 50 12.5 100 50 50 Rhlzopus semi 25 25 25 12. 5 100 100 50 Botrutis cinerea--- 25 25 25 12.5 100 75 50 Byssochlamys fulva- 25 25 25 12. 5 100 75 50 Alternaria Sp.. 50 12.5 75 50 TAB LE IX 3. The process of claim 1 wherein the compound is 2- cinnamyl-4-methoxy-phenol.

Influence of 4-Cinnamyl-phenol on Fermentation of Fruit Juice Inoculated with Yeasts Time for initiation of fermentation, hours Amount of inoeulum, Zero 12.5 p.p.m. 25 ppm. 50 p.p.m. 100 p.p.m. 200 ppm. Run Substrate Inoculum cells/m1. 4-01 of i -P o 4-CP of 4-01 of 4-0? 4-01 1. Gm; e iuice Wild yeast 7 n.d. l2 n.d. 44 n.d. 2 o do 10 17 17 62 96 n.d. 3 0 Wine y 10 12 22 146 w w 4 0 do 10 41 w m m w 5 Apple Juice.... Wine yeast.. 10 46 42 76 a on w .NorE.--Ex lanation' of symbols: 4-01 designates Minnamyl-phenol; n.d. means not determined; on means th t fermentation did not occur, t e organisms had been destroye Having thus described the invention, what is claimed 1. A process for inhibiting the growth of microorganisms selected from the group consisting of bacteria, yeasts, and molds in a substance normally subject to spoilage by said microorganisms, which comprises applying to said substance an efiective microbial-growthhibiting amount of a compound of the structure:

wherein 1 to 3 of the Rs are hydroxy, and the remainder of the Rs are each independently selected from the group consisting of lower alkoxy and hydrogen wherein at least one, of the Rs is lower alkoxy.

2. The process of claim 1 wherein the compound is 2- methoxy-4-cinnamyl-phenol.

References Cited Iurd: Tetrahedron Letter N0. 33, pp. 2863-2866 (1969).

Jurd: Tetrahedron, vol. 25, pp. 1407-1416 (1969).

Jurd: Experientia, vol. 24, pp. 858860 (1968).

Bokadia et al.: vol. 57, pp. 3396-3398 (1962), Chem. Abst.

Mumm et al.: Chem. Abst., vol. 32, pp. 522-524 Hurd et al.: Iour. Am. Chem. Soc., vol. 59, pp. 107- 109 (1937).

Barnes et al.: Tetrahedron, vol. 21, pp. 2707-2715 (1965).

Claisew et al.: Chem. Abst., vol. 19, p. 2038 (1925).

JEROME D. GOLDBERG, Primary Examiner A. I. ROBINSON, Assistant Examiner US. Cl. X.R. 252-404 

